The dopamine D(2) receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle

Dopamine receptors are G protein-coupled receptors that are divided into two subgroups, "D(1)-like" receptors (D(1) and D(5)) that couple to the G(s) protein and "D(2)-like" receptors (D(2), D(3), and D(4)) that couple to G(i). Although inhaled dopamine has been reported to induce bronchodilation in patients with asthma, functional expression of dopamine receptor subtypes has never been described on airway smooth muscle (ASM) cells. Acute activation of G(i)-coupled receptors inhibits adenylyl cyclase activity and cAMP synthesis, which classically impairs ASM relaxation. In contrast, chronic activation of G(i)-coupled receptors produces a paradoxical enhancement of adenylyl cyclase activity referred to as heterologous sensitization. We questioned whether the dopamine D(2)-like receptor is expressed on ASM, whether it exhibits classical G(i)-coupling, and whether it modulates ASM function. We detected the mRNA encoding the dopamine D(2) receptor in total RNA isolated from native human ASM and from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the dopamine D(2) receptor protein in both native human and guinea pig ASM and cultured HASM cells. The dopamine D(2) receptor protein was immunohistochemically localized to both human and guinea pig ASM. Acute activation of the dopamine D(2) receptor by quinpirole inhibited forskolin-stimulated adenylyl cyclase activity in HASM cells, which was blocked by the dopamine D(2) receptor antagonist L-741626. In contrast, the chronic pretreatment (1 h) with quinpirole potentiated forskolin-stimulated adenylyl cyclase activity, which was inhibited by L-741626, the phospholipase C inhibitor U73122, or the protein kinase C inhibitor GF109203X. Quinpirole also stimulated inositol phosphate synthesis, which was inhibited by L-741626 or U73122. Chronic pretreatment (1 h) of the guinea pig tracheal rings with quinpirole significantly potentiated forskolin-induced airway relaxation, which was inhibited by L-741626. These results demonstrate that functional dopamine D(2) receptors are expressed on ASM and could be a novel therapeutic target for the relaxation of ASM.     

Hydrophobic Residues of the D2 Dopamine Receptor Are Important for Binding and Signal Transduction

Abstract: Dopamine receptors belong to the seven transmembrane helix-containing, G protein-coupled receptor superfamily. Mutagenesis studies suggest that dopamine and its analogues interact with aspartate-114 in helix 3 and two helix 5 serines (194 and 197) of the D₂ receptor. In addition to these amino acids, hydrophobic residues within the receptor core may be important not only for binding but also for receptor activation. Described is a site-directed mutagenesis investigation into the roles of these hydrophobic residues in the long isoform of the human D₂ receptor. Replacement of helix 6 phenylalanines (389 or 390) with alanines resulted in disrupted binding to several agonists and antagonists and impaired inhibition of adenylyl cyclase activity. Replacement of the helix 5 phenylalanine-198 with an alanine selectively disrupted [³H]N-0437 binding, whereas the affinities for other agonists and antagonists remained unchanged. This mutant remained functionally intact when stimulated with dopamine or bromocriptine. Replacement of the helix 7 phenylalanine-411 or the helix 6 leucine-387 with alanines produced receptors that bound agonists well but were unable to inhibit adenylyl cyclase. Based on these data, two conserved helix 6 phenylalanines (389 and 390) appear to be crucial for ligand binding, and phenylalanine-411 in helix 7 and leucine-387 in helix 6 may be important for propagating conformational changes from the agonist binding site(s) to G protein coupling domain(s) of the D₂ receptor.     

Functional Importance of the Carboxyl Tail Cysteine Residues in the Human D1 Dopamine Receptor

Abstract: To assess the importance of the cysteine residues Cys³⁴⁷ and Cys³⁵¹ in the carboxylic tail in the human D₁ dopamine receptor, seven mutant receptors were constructed by PCR. The pharmacological and functional properties of the wild-type and mutant receptors were assessed following transient expression in COS-7 cells. Affinities for [³H]SCH 23390 of mutant S347 (Cys³⁴⁷→ Gly), T348 (Tyr³⁴⁸→ stop), S351 (Cys³⁵¹→ Gly), T351 (Cys³⁵¹→ stop), T352 (Pro³⁵²→ stop), and S347/S351 (Cys³⁴⁷→ Gly and Cys³⁵¹→ Gly) were similar to that of wild-type receptor, whereas the expression levels were reduced up to 80%. The potency of dopaminergic antagonists for these mutant receptors was very similar to that of the wild-type receptor. However, mutant T347 (Cys³⁴⁷→ stop) showed a 15–25-fold reduced affinity for the antagonists SCH 23390, (+)-butaclamol, and cis-flupentixol, thus not allowing radioligand analysis. Wild-type and mutant receptors responded dose-dependently with similar potency to dopamine and SKF 38393 with an increased adenylyl cyclase activity. However, mutant receptors with the Cys³⁴⁷ residue changed or removed displayed a diminished ability to activate adenylyl cyclase. Dopamine preexposure desensitized wild-type and mutant S351 receptors. However, mutant receptors with Cys³⁴⁷ replaced or the distal part of the carboxyl tail removed were unable to desensitize. Thus, Cys³⁴⁷ in the cytoplasmic tail of the human D₁ dopamine receptor is important for the receptor in maintaining the conformation for antagonist binding, to play a crucial role in activation of adenylyl cyclase, and to be essential for agonist-induced desensitization.     

Heterologous desensitization of the human dopamine D1 receptor in Y1 adrenal cells and in a desensitization-resistant Y1 mutant.

In previous studies, mutant clones (designated Y1DR) were isolated that resisted ACTH-induced homologous desensitization of adenylyl cyclase. The Y1DR mutation also conferred resistance to the homologous desensitization induced by agonist stimulation of transfected beta 2-adrenergic receptors. These observations suggested that ACTH and beta 2-adrenergic agonists homologously desensitized adenylyl cyclase in Y1 cells by a common mechanism. In the present study, parental Y1 cells (Y1DS) and the Y1DR mutant were transfected with the gene encoding the human dopamine D1 receptor and examined for regulation of adenylyl cyclase by dopaminergic agonists. Transformants were isolated from both cell lines and shown to respond to dopamine agonists with increases in adenylyl cyclase activity. Treatment of the Y1DS transformants with ACTH promoted a rapid, homologous desensitization of adenylyl cyclase and had little effect on the responses to dopamine or NaF; treatment of Y1DS with dopaminergic agonists promoted a slower rate of heterologous desensitization that diminished responsiveness of the adenylyl cyclase system to dopamine, ACTH, and NaF. Y1DR cells transfected with the dopamine D1 receptor were resistant to the heterologous desensitization of adenylyl cyclase induced by dopaminergic agonists. These latter observations suggest that the pathways of homologous desensitization and heterologous desensitization converge at a common point in the desensitization pathway defined by the DR mutation in Y1 cells.     

Inhibition of Adenylyl Cyclase Activity by a Homogeneous Population of Dopamine Receptors: Selective Blockade by Antisera Directed Against Gi1 and/or Gi2

Abstract: The 7315c pituitary tumor cell expresses a homogeneous population of dopamine receptors that are functionally similar to brain dopamine D₂ receptors. [³H]-Sulpiride binding to 7315c cell homogenates was specific and saturable, and K _i values for compounds to compete for these sites were highly correlated with values for the same compounds at D₂ receptors in brain. Dopamine maximally inhibited ∼65% of forskolin-stimulated cyclase activity in cell membranes. Some D₂ agonists had lower efficacies, suggesting that some compounds are partial agonists at this receptor. Removal of GTP from the assay buffer or pretreatment of the tissue with pertussis toxin abolished the inhibition of adenylyl cyclase by dopamine. Immunodetection of most of the known Gα subunits revealed that G_i1, G_i2, G_i3, G_o, G_q, and G_s are present in the 7315c membrane. Pretreatment with the AS antibody (which recognizes the C-terminal regions of Gα_i1 and Gα_i2) significantly attenuated the inhibition of adenylyl cyclase activity by dopamine, whereas antibodies to C-terminal regions of the other Gα subunits had no effect. These findings suggest that the dopamine D₂ receptor regulates cyclase inhibition predominantly via G_i1 and/or G_i2 and that the 7315c tumor cells provide a useful model for studying naturally expressed dopamine D₂ receptors in the absence of other dopamine receptor subtypes.     

Coupling of a cloned rat dopamine-D2 receptor to inhibition of adenylyl cyclase and prolactin secretion.

We have previously described a cDNA which encodes a binding site with the pharmacology of the D2-dopamine receptor (Bunzow, J. R., VanTol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M., Machida, C., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787). We demonstrate here that this protein is a functional receptor, i.e. it couples to G-proteins to inhibit cAMP generation and hormone secretion. The cDNA was expressed in GH4C1 cells, a rat somatomammotrophic cell strain which lacks dopamine receptors. Stable transfectants were isolated and one clone, GH4ZR7, which had the highest levels of D2-dopamine receptor mRNA on Northern blot, was studied in detail. Binding of D2-dopamine antagonist [3H]spiperone to membranes isolated from GH4ZR7 cells was saturable, with KD = 96 pM, and Bmax = 2300 fmol/mg protein. Addition of GTP/NaCl increased the IC50 value for dopamine competition for [3H]spiperone binding by 2-fold, indicating that the D2-dopamine receptor interacts with one or more G-proteins. To assess the function of the dopamine-binding site, acute biological actions of dopamine were characterized in GH4ZR7 cells. Dopamine, at concentrations found in vivo, decreased resting intra- and extracellular cAMP levels (EC50 = 8 +/- 2 nM) by 50-70% and blocked completely vasoactive intestinal peptide (VIP) induced enhancement of cAMP levels (EC50 = 6 +/- 1 nM). Antagonism of dopamine-induced inhibition of VIP-enhanced cAMP levels by spiperone, (+)-butaclamol, (-)-sulpiride, and SCH23390 occurred at concentrations expected from KI values for these antagonists at the D2-receptor and was stereoselective. Dopamine (as well as several D2-selective agonists) inhibited forskolin-stimulated adenylate cyclase activity by 45 +/- 6%, with EC50 of 500-800 nM in GH4ZR7 membranes. Dopaminergic inhibition of cellular cAMP levels and of adenylyl cyclase activity in membrane preparations was abolished by pretreatment with pertussis toxin (50 ng/ml, 16 h). Dopamine (200 nM) abolished VIP- and thyrotropin-releasing hormone-induced acute prolactin release. These data show conclusively that the cDNA clone encodes a functional dopamine-D2 receptor which couples to G-proteins to inhibit adenylyl cyclase and both cAMP-dependent and cAMP-independent hormone secretion.     

多巴胺受体的cDNA克隆

通过多巴胺受体的5个cDNA克隆,综述和分析了5个多巴胺受体(D₁R-D₅R)的基因结构,在染色体上的定位及其mRNA在中枢脑区的分布;比较了这5个受体cDNA克隆的结构特征和药理学性质.     

Cannabinoid Receptor Activation Differentially Regulates the Various Adenylyl Cyclase Isozymes

Abstract: Two cannabinoid receptors belonging to the superfamily of G protein-coupled membrane receptors have been identified and cloned: the neuronal cannabinoid receptor (CB1) and the peripheral cannabinoid receptor (CB2). They have been shown to couple directly to the G_i/o subclass of G proteins and to mediate inhibition of adenylyl cyclase upon binding of a cannabinoid agonist. In several cases, however, cannabinoids have been reported to stimulate adenylyl cyclase activity, although the mechanism by which they did so was unclear. With the cloning of nine adenylyl cyclase isozymes with various properties, including different sensitivities to α_s, α_i/o, and βγ subunits, it became important to assess the signaling pattern mediated by each cannabinoid receptor via the different adenylyl cyclase isozymes. In this work, we present the results of cotransfection experiments between the two types of cannabinoid receptors and the nine adenylyl cyclase isoforms. We found that independently of the method used to stimulate specific adenylyl cyclase isozymes (e.g., ionomycin, forskolin, constitutively active α_s, thyroid-stimulating hormone receptor activation), activation of the cannabinoid receptors CB1 and CB2 inhibited the activity of adenylyl cyclase types I, V, VI, and VIII, whereas types II, IV, and VII were stimulated by cannabinoid receptor activation. The inhibition of adenylyl cyclase type III by cannabinoids was observed only when forskolin was used as stimulant. The activity of adenylyl cyclase type IX was inhibited only marginally by cannabinoids.     

Dopamine disorganizes direct intercellular interactions in keratinocytes cultures: A comparison to hepatocytes

Dopamine in the concentration 0.4 μg/mL abolishes protein synthesis rhythm in HaCaT keratinocytes and hepatocytes unlike noradrenaline or melatonin, which synchronize direct intercellular interactions and organize protein synthesis rhythm. Experiments with D2 dopamine receptors blocking agent metoclopramide (tserukal) in the concentration 2 μg/mL show that a disorganizing effect of dopamine is driven by the activation of D2 receptors, which block adenylyl cyclase and the efflux of calcium ions from internal depos according to the literature. It is shown that tserukal does not activate serotonin receptors in our experimental settings. Cellular interactions’ recovery during or after dopamine action is carried out by melatonin in the concentration 0.001 μg/mL. A recommendation to inject melatonin before dopamine administration for different medical indications is discussed.     

Peptides derived from the third cytoplasmatic loop of serotonin 1B receptor subtype selectively inhibit serotonin signal transduction via a homologous receptor

The third intracellular loops of hormonal receptors play the main role in the interaction of majority of the serpentine type receptors with heterotrimeric G-proteins. In recent years, it was shown that synthetic peptides corresponding to membrane-proximal regions of these loops could be selectively influenced with hormonal signal transduction via the receptors homologous to them and trigger signalling cascade in absence of the hormone. For the first time, we succeeded in synthesizing the peptides derived from C-terminal region of the third intracellular loop of the IB-subtype serotonin receptor and studied their influence on serotonin-sensitive adenylyl cyclase system in the rat brain. The peptides 300-316 and 306-316 (the numbers correspond to amino acid positions in the rat IB-subtype serotonin receptor) at micromolar concentrations in absence of hormone-stimulated GTP-binding of Gi,-proteins coupled with the IB-subtype serotonin receptors and inhibited forskolin-stimulated adenylyl cyclase activity. Using selective agonists and antagonists of serotonin receptors it was shown that the peptides 300-316 and 306--316 inhibited serotonin signal transduction via homologous to them receptor and weakly influenced other types of serotonin receptors. The peptide 300-316 is more active compared with its shorter analogue 306-316 in the selectivity and efficiency of action on adenylyl cyclase signalling system regulated via the IB-subtype serotonin receptors. These findings indicate that the regions 300-316 of the IB-subtype serotonin receptor are involved in interaction with Grproteins and consist of the main molecular determinants responsible for serotonin signal transduction to adenylyl cyclase.     

Stimulated D(1) dopamine receptors couple to multiple Galpha proteins in different brain regions

Previous studies have revealed that activation of rat striatal D(1) dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G(s) and G(q), respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D(1) dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D(1) dopamine receptors couple differentially to multiple Galpha protein subunits. Antisera against Galpha(q) blocks dopamine-stimulated PIP(2) hydrolysis in hippocampal and in striatal membranes. The binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(i) was enhanced in all brain regions. Dopamine also increased the binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(q) in these brain regions: hippocampus = amygdala > frontal cortex. However, dopamine-stimulated binding of [(35)S]GTPgammaS to Galphas only in the frontal cortex and striatum. This differential coupling profile in the brain regions was not related to a differential regional distribution of the Galpha proteins. Dopamine induced increases in GTPgammaS binding to Galpha(s) and Galpha(q) was blocked by the D(1) antagonist SCH23390 but not by D(2) receptor antagonist l-sulpiride, suggesting that D(1) dopamine receptors couple to both Galpha(s) and Galpha(q) proteins. Co-immunoprecipitation of Galpha proteins with receptor-binding sites indicate that in the frontal cortex, D(1) dopamine-binding sites are associated with both Galpha(s) and Galpha(q) and, in hippocampus or amygdala, D(1) dopamine receptors couple solely to Galpha(q). The results indicate that in addition to the D(1)/G(s)/adenylyl cyclase system, brain D(1)-like dopamine receptor sites activate phospholipase C through Galpha(q) protein.     

Pharmacological characterization of dopamine receptors in the corpus allatum of Manduca sexta larvae

Dopamine receptors previously identified in corpora allata (CA) of Manduca sexta last instars on the basis of dopamine effects on JH (juvenile hormone)/JH acid biosynthesis and cyclic AMP (cAMP) accumulation, were characterized pharmacologically. For this study, a broad spectrum of agonists or antagonists of D1, D2, D3 or D4 dopamine receptors, together with the dopamine metabolite N-acetyl-dopamine, other neurotransmitters and their agonists/antagonists, were tested for their effects on gland activity and cAMP production. The lack of effect of other neurotransmitters supports the specificity of the effect of dopamine and the dopamine specificity of the receptors. Only the D2 receptor antagonist spiperone had a potent effect on JH biosynthesis and cAMP formation by CA taken on day 0 of the last stadium, when dopamine stimulates both activities and thus appears to be acting via a D1-like receptor. Several other D2 receptor antagonists, and D1, D2/D1 and D4,3/D2 receptor antagonists were less effective. Thus, the D1-like receptor of the Manduca CA appears to be distinct pharmacologically from vertebrate D1 receptors. By contrast, a number of D2 agonists/antagonists had a significant effect on JH acid biosynthesis and cAMP production by the CA from day 6 of the last stadium, when dopamine inhibits both activities and thus appears to be acting via a D2-like receptor. Certain D1-specific agonists/antagonists were equally effective. The Manduca D2-like receptor therefore bears some pharmacological resemblance to vertebrate D2 receptors. N-acetyl dopamine acted as a dopamine agonist with day 6 CA, the first identified function for an N-acetylated biogenic amine in insects. Dopamine was found to have the same differential affect on the formation of cAMP in homogenates of day 0 and day 6 brains as it did with CA, and in the same concentration range. Dopamine receptor agonists/antagonists affecting cAMP formation by day 0 and day 6 CA homogenates had similar effects with brain homogenates. By contrast, dopamine only stimulated cAMP formation by homogenates of day 0 and day 6 abdominal or ventral nerve cord. These results suggest that D1- and D2-like dopamine receptors of Manduca are regionally as well as temporally localized.     

D2 dopaminergic receptors: normal and abnormal transduction mechanisms.

Dopamine receptors of D2 type present on lactotroph cells are coupled to a large series of transduction mechanisms. Beside their negative coupling with adenylate cyclase, they are also coupled with potassium and calcium channels, leading to a decreased intracellular calcium concentration. In addition, D2 dopamine receptors also modulate phospholipase activities. Dopamine inhibits inositol phosphate production, through two distinct mechanisms. One of them could represent a direct negative coupling with phospholipase C. All these transduction mechanisms of the D2 dopamine receptors implicate G proteins sensitive to pertussis toxin. In contrast, these receptors are negatively coupled to phospholipase A2 through G proteins insensitive to this toxin. Both isoforms of the D2 dopamine receptor, generated by alternate splicing of a single gene, are present in lactotroph cells. After transfection in CH4C1 cells the two isoforms are coupled with adenylate cyclase while only the shortest isoform appears negatively coupled to phospholipase C. Functional D2 dopamine receptors are present in human prolactinomas. Resistance to bromocriptine therapy is associated with a decreased density of these receptors in the tumor. In addition, the ratio of the two receptor isoforms (measured by PCR) is different in responsive and resistant tumors. Furthermore, the activity of Gi/Go proteins coupled to adenylate cyclase appears also affected in resistant tumors. Resistance to bromocriptine therapy appears thus to involve multiple changes at the different levels of the multiple mechanisms of action of dopamine on lactotroph cells.     

Studies of dopamine pharmacology in molluscs

Dopamine has been established as a putative neurotransmitter in several species of molluscs. Biochemical and neurophysiological studies of the cellular pharmacology of dopamine have revealed several properties of molluscan dopamine receptors. The biochemical synthesis and degredation of dopamine in molluscs follows the same pathways that have been described in mammals. Adenylate cyclase is present, and the receptor mediating CAMP production is blocked by neuroleptics and certain ergot alkaloids. Studies of this enzyme and of radioligand binding indicate that molluscan dopamine receptors and serotonin receptors share certain characteristics. Neurophysiological studies have shown that dopamine induces several forms of ionic conductance changes in molluscan neurons. The receptors mediating these conductance changes may be differentiated pharmacologically. Neuroleptics are antagonists at certain receptors and ergot alkaloids have been shown to be either partial agonists or antagonists. Present evidence indicates that molluscan and mammalian CNS dopamine receptors have some similarities. However, further biochemical and neurophysiological investigations will be necessary to fully characterize molluscan dopamine receptors.     

Dopamine receptors in the goldfish retina: 3H-spiroperidol and 3H-domperidone binding; and dopamine-stimulated adenylate cyclase activity

Dopamine receptors in the goldfish retina have been examined by binding studies using ³H-spiroperidol and ³H-domperidone as specific ligands, and by measuring retinal adenylate cyclase activities in the presence and absence of dopamine. Our results indicate that washed membranes from goldfish retinal homogenate bind a variety of dopamine agonists and antagonists with high affinities and with characteristics similar to those reported for the brain, with the exception that in this retina there is virtually no binding of the specific D₂ receptor antagonist, ³H-domperidone. In addition, there is a very low basal activity of adenylate cyclase which can be greatly stimulated by dopamine, possibly reflecting a high degree of coupling between this enzyme and the dopamine receptor. Taken together, our findings indicate that the goldfish retina contains a high density of D₁ type dopamine receptors and few, if any, D₂ type receptors.     

A comparison of the properties of the cardiac beta-adrenergic receptor adenylyl cyclase system in the rat and the marmoset monkey

1. A comparison was made between adrenergic receptor binding properties and catecholamine-stimulated adenylyl cyclase activity in cardiac membrane fractions from the rat and the marmoset monkey. 2. [125I]HEAT and [125I]ICYP were used to determine respectively, the alpha- and beta-adrenergic receptor binding in cardiac membrane fractions. 3. Greatest adrenergic receptor density and degree of specific binding was evident using membranes sedimenting between 6000 and 46,000 g. 4. In rat heart, the ratio of beta- to alpha-adrenergic receptors was 57:43, while for the marmoset this ratio was 92:8. 5. Basal, isoproterenol, sodium fluoride and forskolin-stimulated adenylyl cyclase activities in the rat and marmoset monkey were investigated in several different cardiac membrane fractions. 6. The highest-fold stimulation of adenylyl cyclase activity was present in membranes sedimenting between 0 and 500 g. 7. Adenylyl cyclase activities were higher in the marmoset heart membrane preparations, however the rat heart adenylyl cyclase exhibited greater sensitivity to isoproterenol; ED50 3.8 X 10(-7) M compared with 7.5 X 10(-7) M for the marmoset. 8. Differences between rat and marmoset catecholamine-sensitive adenylyl cyclase activity were apparent when a variety of adrenergic agonists and antagonists were tested. 9. In the marmoset but not the rat, adrenergic antagonists alone stimulated basal adenylyl cyclase activity. 10. Differences in the activation of cardiac adenylyl cyclase by GTP and GMP-PNP were also evident between the rat and the marmoset monkey, particularly with regard to basal and isoproterenol-stimulated activity.     

Synthesis and characterization of selective dopamine D2 receptor antagonists

A series of indole compounds have been prepared and evaluated for affinity at D2-like dopamine receptors using stably transfected HEK cells expressing human D2, D3, or D4 dopamine receptors. These compounds share structural elements with the classical D2-like dopamine receptor antagonists, haloperidol, N-methylspiperone, and benperidol. The compounds that share structural elements with N-methylspiperone and benperidol bind non-selectively to the D2 and D3 dopamine receptor subtypes. However, several of the compounds structurally similar to haloperidol were found to (a) bind to the human D2 receptor subtype with nanomolar affinity, (b) be 10- to 100-fold selective for the human D2 receptor compared to the human D3 receptor, and (c) bind with low affinity to the human D4 dopamine receptor subtype. Binding at sigma (sigma) receptor subtypes, sigma1 and sigma2, were also examined and it was found that the position of the methoxy group on the indole was pivotal in both (a) D2 versus D3 receptor selectivity and (b) affinity at sigma1 receptors. Adenylyl cyclase studies indicate that our indole compounds with the greatest D2 receptor selectivity are neutral antagonists at human D2 dopamine receptor subtypes. With stably transfected HEK cells expressing human D2 (hD2-HEK), these compounds (a) have no intrinsic activity and (b) attenuated quinpirole inhibition of adenylyl cyclase. The D2 receptor selective compounds that have been identified represent unique pharmacological tools that have potential for use in studies on the relative contribution of the D2 dopamine receptor subtypes in physiological and behavioral situations where D2-like dopaminergic receptor involvement is indicated.     

Identification of D1-like dopamine receptors on human blood platelets

Dopamine is able to inhibit the epinephrine-induced aggregation of human blood platelets, but the mechanism of action has not been elucidated. In this study we report that membranes from human blood platelets possess high affinity, saturable and stereoselective binding sites for the D1 dopamine receptor antagonist (3H) SCH 23390. (3H) SCH 23390 appeared to label a single class of binding sites with a Bmax of 18.6 +/- 1.6 fmol/mg protein and a KD of 0.8 nM. The potencies of different dopaminergic antagonists and agonists in displacing (3H) SCH 23390 from blood platelet membranes were similar to those obtained for striatal membranes. Unlike the classically defined D1 receptors, e.g. those in striatum, the D1 receptor sites on platelets appeared not to be coupled to the adenylate cyclase system, hence the term "D1-like". The D1 agonist SKF 38393 was more potent than dopamine in inhibiting platelet aggregation induced by epinephrine, and the effects of dopamine and SKF 38393 were prevented by SCH 23390. These results suggest that the inhibitory action of dopamine on the epinephrine-induced platelet aggregation is mediated through these D1-like receptors.     

The dopamine of the rat mammotroph in cell culture as a model for drug action

Dopamine can act directly on pituitary cells to inhibit prolactin release. This action can be blocked by dopamine receptor blocking drugs such as haloperidol, sulpiride and other neuroleptic agents. Comparison of the properties of the mammotroph dopamine receptor with the adenylate cyclase linked dopamine receptor of the limbic forebrain reveals some obvious differences. For example, dopamine receptor stimulants such as S-584 and lergotrile mesylate are inactive in stimulating the adenylate cyclase preparations but are potent in inhibiting pituitary prolactin secretion. Such inhibition of prolactin secretion can be reversed by haloperidol or sulpiride. In contrast to these observations, sulpiride does not block dopamine stimulation of cAMP formation. In addition, dopamine, apomorphine or lergotrile mesylate have no effect on a pituitary adenylate cyclase preparation and dopamine fails to elevate cAMP in the intact cells in culture. Despite the similarity between these two dopamine sensitive systems with respect to a number of agonists and antagonists, the exceptions described suggest that the pituitary system with further study may offer some greater reliability as a predictive test for clinically useful agents. These results also suggest that the receptors for dopamine, like that for norepinephrine, are of two types, only one of which is coupled to adenylate cyclase.